Apparatus for the cultivation of microorganisms



Aug. 17, 1954 MONQD 2,686,754

APPARATUS FdR THE CULTIVATION OF MICROORGANISMS Original Filed Nov. 28,1950 2 Sheets-Sheet 1 k1 ll tic 01/55. W n

Aug. 17, 1954 J MQNQD 2,686,754

APPARATUS FOR THE CULTIVATION OF MICROORGANISMS Original Filed Nov. 28,1950 2 Sheets-Sheet 2 IIVI/ENTUIP I? (in C1905: mom-0a Ma, zi/

Patented Aug. 17, 1954 APPARATUS. FOR THE CULTIVATION F MICRQQRGANISMSJacques Monod,,Paris, France, assignor to Le Bactogene}? S. A. It. L.,Paris, Seine, France, a society of France Original application November28, 1950, Serial No.

197,867. Divided and this application February g6, 1952, Serial No.270,125

Thepresent invention relates to apparatus for (bacteria,

the cultivation of microorganisms yeastsymoulds, protozoa, lower algae,etc).

Its chief object is to achieve a continuous production of the desiredculture with an adjustable output and ahigh yieldwhile maintainingconstancy and homogeneityof the qualities of the product. 1 l

Embodiments of my invention will be hereinafter described with referenceto the accompanyj ing drawings, given merely-byway of example,

and in which:

Fig. 1 is a diagrammatic sectional view of an apparatus for theobtainment of microorganism cultures according to my invention; Fig. 2is a similar view of an apparatus for the V same purpose made accordingto another embodif ment;

Fig. 3 is a detail view illustrating a feature o-f zo For the sake ofclarity, a will first be reminded that I start from a culture brought tothe demy invention.

sired density in a suitable container, in the presence of a suitablegaseous phase, and I continue ously add fresh nutrient medium theretoand continuously remove preparedculture therefrom at the same rate,while subjecting the culture in the course of evolution to an action(forinstancey a stirring one) capable of keeping it homogeneous, that isto say equally rich in cells in all of its parts.

The rateat which fresh medium is added and culture is removed is equalto or lower than a limit which conditions the maintainin of the densityor richness in cells of th culture, at a stable level of equilibrium.

Furthermore, the possibilities of interchange between the gaseous phaseand the liquidphase are made asiclose as possible to theconditions;

of equilibrium between these two phases and in saturation equilibriumbetween the oxygen of air and the dissolved oxygen.

5 Claims. 3 (01. 195-143) overpressure may eventually be provided,' oipose sibly a partial vacuum.

The gaseous atmosphere may be introduced'at the beginning of theoperations and remain for the Whole duration of the treatment; or itmay,

on the contrary, be renewed, either in a continu ousor in adiscontinuous manner.

The treatment container consists of a rotary" cylinder or drum, inparticular o fhorizontal or substantially horizontal axis, thisarrangement ensuring both the liquid stirring action and the formationof liquid films constituted inthis case by the wet surfaceso-f thecontainer, which are:

constantly renewed.

At least one of the two above mentioned actions (stirring and filmformation) may becom pleted by suitable means.

For instance, as hereinafter described with j ref-"T erence to Fig. 1,by a spiral rib or piece rotating in the cylinder; and/or (Fig. 2) byone or several cylinders disposed inside the first one; and/or by plates"of any suitable shape having relative move ments of -immersion, thenextraction, with'respect to the culture liquid; or by static devices in-Concerning the actionof the gaseous phase, 1

will be achieved or intensified, preferably, by oreating in thetreatment container large inter? change areas constituted by liquidfilms in move; ment and quickly renewed on suitable metallic:

or other surfaces.

Accordingly; the gases will be preferably introduced without beinginjected into the liquid. An

eludin for instance inclined planes on which the liquid is caused toflow. i

Finally, means are provided, if necessary, for maintaining a giventemperature in the appa ratus, which means may consist in heatingresistors or include the preliminary heating of the fluids intended topass through the apparatus,

etc.

i Fig. 1 shows an apparatus including a cylindrical tank and complyingwith the above conditions.

It is essentially constituted by a horizontal or substantiallyhorizontal drum, provided at its end with two trunnions 2 and 3 restingin ball bearzings 4 and 5. This drum is given a rotary move- 40particular to the conditions for maintaining ment through a pulley 6driven by a motorl.

the operation of the apparatus enter the cylinder through discs 8 and 9in which they are fitted with fluidtight joints.

The inner wall of the drum is provided mspiral-shaped rib It the pitchand height of which are determined by the type of operation thatisconsidered. Heating at constant temperature is obtained either by placinthe drum in a' thermostatic vessel Or by disposing electric re sistanceson'the outer surface or evenonthein side of the drum (or through anyother means).

delivery pump or any other adequate device. The

device further includes a gas inflow tube [4 (for air or any othersuitable gaseous mixture, sterilized by heating or filtering) and anoutlet tube I5. Other tubes, such as a special pipe is for inoculation,may be provided.

I may even, if so desired, provide inside the apa to the inside.

paratus a source of radiation (infra-red, ultraviolet, X-rays, etc).

It should be noted that, in the embodiment shown, the level H of theliquid (culture in treatmeant) is higher than the height h of the spiralrib It. It is also visible that the liquid is caused to circulateconstantly in a closed circuit along the path indicated by arrows fifz.The desired stirring action is thus obtained, which ensures homogeneityof the culture in all of its parts, despite the addition of liquid at 2and the removal of culture at l3.

In the embodiment of Fig. 2, cylinder l is provided with coaxialcylinders, having continuous or perforated surfaces, such as 22, '23, oflengths and diameters smaller than those of the drum, to which they maybe secured by braces 24, 25, 2E, 21. But they might turn, independentlyof said drum, possibly in the opposite directions, being driven throughmechanisms (for instance concentric shafts) such as diagrammaticallyshown in dotted lines (reference characters IBI to I04). In this case,the above mentioned braces, instead of fixing the cylinders with respectto one another would serve only to keep them centered with respect toone another. They may also be providedwith helical or other ribsanalogous to rib In (Fig. 1) to ensure a thorough stirring. Eventually,drum i might be stationary and only cylinders 22, 23, rotary, anarrangement which might facilitate the inflow and outflow of the liquidsand gases. Anyway, as they are open at both ends (so as to enable air orgases to circulate freely therethrough) and partly immersed, cylinders22, 23 increase the areas of interchange between the liquid and gaseousphases.

In, some cases, and in particular in that of apparatus of great size, itis of interest to provide as high as possible a bacteriologicalsterility, through suitable means. These means may be constituted, inparticular, by bafiies provided in the apparatus bearings and providedwith sterilizing devices such as ultra-violet ray tubes receiving ashape adapted to the desired purpose. Thus, although these bearings arenot perfectly gastight, sterility is maintained since the gases whichmay enter through the joints are sterilized.

On Fig. 3, which illustrates one embodiment of such means, the end oftrunnion 3 carries a coaxial cylindrical element I40, provided with ahole in its base 150. Between cylinders 3 and Mn there is provided acylindrical element I60 rigid with closure plate 9, which is providedwith a flange no. On either side of cylinder I50 are provided mercuryvapour tubes I80, for instance spiralwound, which are fed with currentfrom 4 an external source by means of terminals, not shown on thedrawing, fixed to the closure plate. Any air which may enter along thepath indicated by the arrows is therefore necessarily subjected to theultra-violet radiation and therefore sterilized.

Closure plate!) is fixed. It is, for instance, rigid with the apparatusframe or bearing 5 on which rest the rotary drums. No mechanical contactexists between the elements of the trunnion and those of the closureplate.

In an apparatus such as those shown by way of example for carrying outthe invention, a slight overpressure may be provided in the container,which makes it possible to prevent ozone formed by the ultra-violet raysfor instance, to penetrate The atmosphere might then become toxic. Itsuflices, in order to obtain this overpressure, suitably to adjust theflow rates through conduits such as I4 and [5 (Fig. 1).

The operation of such an apparatus, for instance that of Fig. 1, takesplace. as follows:

Drum I is first filled with fresh nutrient medium up to a levelcorresponding to the volume V which it is desired to maintainpermanently in the apparatus, and therefore to the desired output perhour. A sterilization is performed bysteam or other suitable means, orby making use of the thermostat heating device. i

Once the respective tubes have been connected and the temperature iscorrect, the medium is -tinuous manner by feeding fresh liquid at J2 Inthe.

inoculated in a sterile manner through tube [6 and the motor is started.The speed of this motor will be chosen so as to obtain an efficient andquick stirring. Stirring is achieved in the plane perpendicular to theaxis through rotation it-,

self and in the plane of the generatrices by spiral rib In. Equilibriumbetween the gaseous and liquid phases will be further ensured by thelarge area of interchange constituted by the liquid surface and the wetsurfaces of the. drum and spiralrib. Growth of the culture is thenstudied by collecting samples at time intervals until the.

desired density is obtained.

From this time on, production goes on in a conaccording to the desiredoutput rate. embodiment shown by the drawing, siphon 13 suppliesanoutput of culture at the rate of the feed of fresh medium. v If ratioD is lower than the above mentioned limit or nearly equal to this limit,the apparatus supplies a culture which is perfectly homogeneous and thedensity of which remains constant, both duction per hour may be modifiedat any timeand given the desired value by modifying volume V, that is tosay the level of the liquidin the apparatus and modifying the outputrate accordingly, the ratio D remaining the same or still complying withthe same condition. I might also modify the output rate withoutmodifying volume V.

In a general manner, the rate of growthwill automatically becomeadjusted in accordance with the output rate whereby my method makes itpossible to adjust the rate of growth of the culture and to give it anyvalue lower than or nearly equal to the maxi mum rate of growth of themicro-organism that is considered in the medium used, i. e. to fix anddetermine one of the essential characteristics of growth, upon whichmost of the qualities and properties of the product depend. My methodtherefore truly makes it possible to obtain adjustment at will of therate of growth.

Of course, while operating in a continuous manner, I might modify atwill, for given periods of time, the rates of output.

In a general manner, while I have, in the above description, disclosedwhat I deem to be praotical and eificient embodiments of my invention,it should be well understood that I do not wish to be limited thereto asthere might be changes made in the arrangement, disposition and form ofthe parts without departing from the principle of the present inventionas comprehended within the scope of the accompanying claims.

What I claim is:

1. An apparatus for the obtainment of a microorganism culture in liquidform which comprises, in combination, a cylindrical drum ofsubstantially horizontal axis to contain the liquid culture intreatment, at least one other drum open at both ends mounted in saidfirst mentioned drum coaxially therewith, the first mentioned drumhaving end walls extendin continuously, in the vertical plane of saidaxis, from the lowest generatrix of said first mentioned dllllll to alevel higher than the level of the lowest generatrix of the secondmentioned drum, whereby liquid may be ltept in the first mentioned drumat a level such that the second mentioned drum constantly dips in thisliquid, means for rotating at least one of said drums, means forcontinuously feedin fresh nutrient medium into said first mentioned drumat one end thereof, and means for continuously removing culture fromsaid first mentioned drum at the other end thereof.

2. An apparatus for the obtainment of a microorganism culture in liquidform which comprises,

coaxially therewith, the first mentioned drum having end walls extendingcontinuously, in the vertical plane of said axis, from the lowestgeneratrix of said first mentioned drum to a level higher than the levelof the lowest generatrix of the second mentioned drum, whereby liquidmay be kept in the first mentioned drum at a level such that the secondmentioned drum constantly dips in this liquid, means for rotating atleast one of said drums, means for continuously feeding fresh nutrientmedium into said first mentioned drum at one end thereof, means forcontinuously removing culture from said first mentioned drum at theother end thereof, and a helical rib carried by said rotating drum forstirring said liquid culture.

3. An apparatus for the obtainment of a microorgaism culturein liquidform which comprises, in combination, a cylindrical drum of substan:tially horizontal axis to contain the liquid structure in treatment, atleast one other drum, open at both ends, mounted in said first mentioneddrum coaxially therewith, the first mentioned drum having end wallsconstituted by annular inward flanges of an inner diameter smaller thanthe diameter of the second mentioned drum, whereby liquid may be kept inthe first mentioned drum at a level such that the second mentioned drumconstantly dips in said liquid, cylindrical hollow trunnions rigid withsaid first mentioned drum extending outwardly from the inner edges ofthe end walls of said first mentioned drum, bearings for supporting saidtrunnions, fixed plates located opposite the outer ends of saidtrunnions, cooperating baifie means carried by said plates and saidtrunnions to restrict communication between the inside of said firstmentioned drum and the external atmosphere, conduit means extendingthrough one of said fixed plates for continuously adding fresh nutrientmedium into said first mentioned drum, conduit means extending throughthe other of said plates for continuously removing culture from saidfirst mentioned drum, a helical rib carried by the inner wall of saidfirst mentioned drum for stirring said liquid culture therein, and meansfor rotating said first mentioned drum about its axis.

4. An apparatus according to claim 3 -further including a mercury vapourtube wound helically in the space between said baflle means forsterilizing gas circulating through said space.

5. An apparatus for the obtainment of a micro-organism culture in liquidform which comprises, in combination, a cylindrical drum ofsubstantially horizontal axis to contain the liquid structure intreatment, at least one other drum, open at both ends, mounted in saidfirst mentioned drum coaxially therewith, the first mentioned drumhaving end walls constituted by annular inward flanges of an innerdiameter smaller than the diameter of the second mentioned drum, wherebyliquid may be kept in the first mentioned drum at a level such that thesecond mentioned drum constantly dips in said liquid, cylindrical hollowtrunnions rigid with said first mentioned drum extending outwardly fromthe inner edges of the end walls of said first mentioned drum, bearingsfor supporting said trunnions, fixed plates located opposite the outerends of said trunnions, cooperating bafile means carried by said platesand said trunnions to restrict communication between the inside of saidfirst mentioned drum and the external atmosphere, conduit meansextending through one of said fixed plates for continuously adding freshnutrient medium into said first mentioned drum, conduit means extendingthrough the other of said plates for continuously removing culture fromsaid first mentioned drum, conduit means extending through one of saidfixed plates for feeding gas under pressure into said first mentioneddrum, to maintain therein a pressure slightly above the pressure in theexternal atmosphere, a helical rib carried by the inner Wall of saidfirst mentioned drum for stirring said liquid culture therein, and meansfor rotating said first mentioned drum about its axis.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 888,685 Arnold May 26, 1908 1,271,433 Cassel July 2, 19182,137,051 Jain Nov. 15, 1938 2,189,279 Bitner Feb. 6, 1940 2,248,526Francois July 8, 1941

1. AN APPARATUS FOR THE OBTAINMENT OF A MICROORGANISM CULTURE IN LIQUIDFORM WHICH COMPRISES, IN COMBINATION, A CYLINDRICAL DRUM OFSUBSTANTIALLY HORIZONTAL AXIS TO CONTAIN THE LIQUID CULTURE INTREATMENT, AT LEAST ONE OTHER DRUM OPEN AT BOTH ENDS MOUNTED IN SAIDFIRST MENTIONED DRUM COAXIALLY THEREWITH, THE FIRST MENTIONED DRUMHAVING END WALLS EXTENDING CONTINUOUSLY, IN THE VERTICAL PLANE OF SAIDAXIS, FROM THE LOWEST GENERATRIX OF SAID FIRST MENTIONED DRUM AT A LEVELHIGHER THAN THE LEVEL OF THE LOWEST GENERATRIX OF THE SECOND MENTIONEDDRUM, WHEREBY LIQUID MAY BE KEPT IN THE FIRST MENTIONED DRUM AT A LEVELSUCH THAT THE SECOND MENTIONED DRUM CONSTANTLY DIPS IN THIS LIQUID,MEANS FOR ROTATING AT LEAST ONE OF SAID DRUMS, MEANS FOR CONTINUOUSLYFEEDING FRESH NUTRIENT MEDIUM INTO SAID FIRST MENTIONED DRUM AT ONE ENDTHEREOF, AND MEANS FOR CONTINUOUSLY REMOVING CULTURE FROM SAID FIRSTMENTIONED DRUM AT THE OTHER END THEREOF.